Retroviral gene expression is a complex process dependent on a multitude of host factors. To meet the unique challenge of producing unspliced, singly spliced, and multiply spliced RNAs from a compact proviral genome, HIV-1 and other retroviruses have evolved elaborate mechanisms to exploit host cell mRNA processing and export pathways. The aim of this proposal is to investigate mechanisms by which specific RNA elements direct the biogenesis, export, and cytoplasmic fates of HIV-1 transcripts. Recently, an N- terminal fragment of hnRNP U (designated N86-hnRNPU) was identified in the Goff laboratory as a potent and specific inhibitor of HIV-1 gene expression. The finding that N86-hnRNPU causes transcripts harboring HIV 3'LTR sequence to be retained in the nucleus suggests that the HIV 3'LTR contains RNA sequences that confer unique requirements for correct transcript biogenesis and/or nuclear export. The mechanism of this inhibition will be investigated using a combination of cell biological and biochemical methods. The subnuclear localization of transcripts targeted by N86-hnRNPU will be used to diagnose the precise step of mRNA maturation affected, allowing a hypothesis-driven investigtion of HIV mRNA processing and RNP assembly. The specific properties of the 3'LTR leading to N86-hnRNPU-sensitivity are unknown. To better understand the ways in which 3'LTR sequences direct RNA processing and assembly events, proteins associated with the HIV 3'LTR will be identified using an RNA-based method for purification of endogenously assembled RNP complexes. This approach will also be used to purify RNPs dependent on the HIV Rev protein for efficient nuclear export, generating further insight into the coupling of RNP assembly and export. Novel HIV mRNA-associated proteins will be overexpressed or depleted by RNAi to determine their roles in retroviral gene expression. A series of assays will be used to test the effects of protein overexpression and depletion on HIV mRNA stabiliity, localization, and translation. Together, these experiments are designed to generate mechanistic insights into HIV biology and identify novel host factors required for viral propagation. Public HealthRelevance: The causative agent of AIDS, the human immunodeficiency virus type 1 (HIV-1) depends on human proteins and cellular processes to create infectious viral particles. This research is intended to identify new human proteins necessary for viral gene expression, and, by extension, viral infectivity. Proteins involved in these processes represent attractive targets for therapeutic intervention.